1. Field of the Invention
Enzyme immunoassay methods are known for the determination of the presence in a sample of an analyte. The known method comprises determining the effect that the sample containing the analyte has on the binding between a conjugate of the analyte (ligand) and an enzyme and a receptor for the analyte. When the conjugate and the receptor bind, a modulation of the enzymatic activity occurs. The presence of analyte in the sample can be determined from the effect that the analyte has on the modulation of the enzymatic activity when compared to that obtained in the absence of analyte or in the presence of known amounts of analyte.
In some cases, the number of molecules of analyte to the number of molecules of enzyme label will be greater than one and in other cases, the number of molecules of enzyme label to number of molecules of analyte in the conjugate will be greater than one. The latter situation may arise where the analyte has a high molecular weight, either because it is a high molecular weight material or because of aggregation of lower molecular weight materials producing a material with a high molecular weight. For example, there are high molecular weight proteins which have drawn increasing attention in recent years as knowledge of their functions and their significance in medical diagnosis has grown. The need for sensitive and efficient assays for these proteins has likewise grown. To this end, various immunoassay techniques have been applied.
The large size of these proteins, however, has heretofore limited the number of immunoassays which could provide a detectable signal. In particular, enzyme immunoassays have been limited to cumbersome procedures involving progressive binding reactions and phase separation. This is a serious limitation since enzyme immunoassays have the advantage of permitting spectrophotometric determinations and the potential of offering high sensitivity due to rapid substrate turnover rates which amplify the signal.
For a large number of analytes, the concentration range of interest will fall between 100 micrograms to 1 pg per ml. For many analytes, the concentration range of interest will vary from about twofold to one hundred fold so that a quantitative determination will require the ability to distinguish small differences in the concentration of the analyte in the assay medium. Immunoassays are predicated on detecting the complexation between the ligand and receptor. The lower the concentration of analyte, the fewer the number of complexes which are formed.
One problem which may arise in enzyme immunoassays is background interference which tends to obscure the modulation of enzymatic activity normally causing a diminution of enzyme activity. Immunoassays of enhanced sensitivity, therefore, are needed particularly those which will permit the determination of large proteins in a simple and efficient manner.
2. Description of the Prior Art
A novel biological assay method for determining the presence of a specific organic material by employing a modified enzyme for amplification is disclosed in U.S. Pat. No. 3,817,837. In U.S. Pat. No. 4,267,270, there is described a method for the determination of antigens and antibodies using site deactivating media.
Solid phase sandwich enzyme immunoassays for ferritin, a protein with a molecular weight of approximately 450,000 daltons, are disclosed in Therialt, et al., Clin. Chem., 23/11:2142-2144 (1977); Fortier, et al., Clin. Chem., 25/8:1466-1469 (1979); Conrade, et al., S. Afr. Med.. J., 57:282-287 (1980); Paige, et al., Scand. J. Clin. Lab. Invest., 40:641-645 (1980); Anderson, et al., Clin. Kim. Acta., 116:405-408 (1981); and Linpisarn, et al., Am. Clin. Biochem., 18:48-53 (1981).
Immunoassays for ferritin which do not involve enzymes include two site immunoradiometric assays as disclosed in Addison, et al., J. Clin Pathol., 25:326-329 (1972) and Miles, et al., Anal. Biochem., 61:209-224 (1974). Competitive radioimmunoassays as disclosed in Porter, J. Lab. Clin. Med., 83:147-152 (1974) and electroimmunoassays as disclosed in Carmel, et al., Anal. Biochem., 85:499-505 (1978) and Laurell, Anal. Biochem., 15:45-52 (1966). Copending application U.S. Ser. No. 258,848 filed Apr. 29, 1981, discloses conjugates of a ligand and .beta.-D-galactosidase for enzyme immunoassays.